This research proposal aims to elucidate the mechanisms for how gap junctions and cytoskeletal components coordinately regulate fiber cell assembly and cell surface interlocking structures to achieve fundamental optical and biomechanical properties of the lens. Lens fiber cell interlocking structures provided by membrane/cytoskeletal structures are essential for lens hemostasis, elasticity and transparency. Gap junctions in lens fiber cells predominantly consist of Cx46 (Gja3) and Cx50 (Gja8) connexins in human and mouse lenses. Gja3 and Gaj8 mutations cause various types of cataracts in humans and mice. Cataractogenesis caused by these connexin mutations is associated with dysfunctional gap junction channels, impaired lens homeostasis and degradation of crystallin proteins. However, the molecular and cellular mechanisms underlying the sequential steps of these pathological events are not well understood. It is unknown how connexin gene mutations lead to various types of cataracts, especially when an identical gene mutation leads to different types of cataracts in human individuals and in different mouse strain backgrounds. Our recent results show that surface tongue-and-groove structures are eliminated in lens mature fibers of Gja3(-/-) lenses, corresponding to nuclear cataracts. Cytoskeletal proteins including CP49 and periaxin act as genetic modifiers to modulate cataract severity of Gja3(-/-) mice in different strain backgrounds. In contrast, a loss of Cx50(Gja8) impairs the ball-and-socket structures of peripheral differentiating fiber cells to delay the fiber cell elongation nd disrupt lens homeostasis to lead to smaller lenses. Specific aims of this project are designed to test our hypothesis that functional coordination between gap junctions and cytoskeletal components controls lens fiber-to-fiber interlocking structures, including protrusions on the short sides and ball-and-sockets on the long sides of hexagonal shaped fiber cells in lens cortex and the tongue/groove structures in mature fiber cells in the lens core. Specific aim 1 will study how Cx46 (Gja3) gap junctions, CP49 and periaxin coordinately control fiber cell surface interlock structures to maintain lens integrity, homeostasis and transparency. We will further identify protein components associated with Cx46 (Gja3) gap junctions/cytoskeleton that are important for maintaining interlocking structures such as protrusions. Specific aim 2 aims to elucidate the molecular basis for how Cx50 (Gja8) gap junctions establish ball-and-socket structures on the long sides of hexagonal shaped fiber cells needed for lens growth. We will identify specific PDZ-domain containing proteins that bind to the C-terminal ends of Cx50 and link to cytoskeleton for establishing ball-and-socket structures. Specific aim 3 will identify and characterize the third genetic modifier on Chromosome 2, which can suppress the effect of 129-periaxin during cataractogenesis. We will further determine specific biochemical, physiological and biomechanical changes associated with nuclear cataracts.